Nucleic acid analysis is a basic clinical and research tool in genetics, cancer and infectious disease. The polymerase chain reaction (PCR) is often used to amplify sequences before analysis. Continuous fluorescence monitoring of rapid cycle PCR combines amplification and analysis for results within 30 minutes. Our objective is to extend the utility of fluorescence rapid cycle PCR by color and Tm multiplexing. A novel sequence-specific fluorescent probe system based on resonance energy transfer will be used for monitoring PCR. A 4- color instrument will be built to test newly available acceptor dyes. The optics and software compensation for fluorescence crosstalk between channels will be adapted from flow cytometry. The melting temperature or Tm of a probe depends on the stability of the duplex formed. Fluorescence melting curves will be obtained as the temperature passes through the Tm. This "dynamic dot blot" differentiates multiple targets by Tm and allows an additional dimension for multiplexing. We will test the feasibility of simultaneous color and Tm multiplexing. The targets to be analyzed will be apolipoprotein E genotyping and mutations in exon 1 of the beta-globin gene. Multiplexing in solution with fluorescent probes and rapid cycle PCR increases the efficiency and speed of complex nucleic acid analysis. PROPOSED COMMERCIAL APPLICATION: Idaho Technology manufactures the RapidCycler(TM) (30 cycles in 15 minutes) and the LightCycler(TM) (30 cycles in 15 minutes with fluorescence monitoring) and has licensed this technology to Boehringer Mannheim (now Roche Molecular Biochemicals). Multiplexing by color and Tm are powerful tools that will be widely used in academics, clinical medicine and the biotechnology industry.